Process for stabilizing the activity of enzymes with phosphorus compounds

ABSTRACT

The invention relates to processes for stabilizing the activity of an enzyme, comprising mixing a phosphine or phosphite with an oxidoreductase enzyme.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of and claimspriority to U.S. patent application Ser. No. 09/522,872, filed Mar. 10,2000, and also claims priority to U.S. Provisional Patent ApplicationSerial No. 60/123,833, filed Mar. 11, 1999, both of which are herebyincorporated by this reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to methods for stabilizing enzymes.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to catalytic processes for the useof H₂ as a reducing agent for organic compounds, in the presence ofcatalysts containing enzymes. Many man-made catalysts are known forreduction and/or hydrogenation reactions, but there are many limitationsin the ability of the known catalysts to selectively reduce orhydrogenate one unsaturated functional group in the presence of anotherfunctional group. Moreover, most prior art catalysts and processescannot selectively produce optically active products, as is highlydesirable in the production of compositions for human or animalconsumption, such as food or pharmaceuticals.

[0004] In contrast, many enzymes are capable of highly selectivereduction of their natural substrates. In some cases enzymes catalyzeunique transformations that require multiple steps by traditionalsynthetic methods. Moreover, a wide range of unnatural substrates,including a wide variety of unsaturated organic compounds, can beenzymatically reduced, with high chemo-, regio- and/orenantioselectivity, under mild reaction conditions. It is thereforehighly desirable to employ enzymatic processes for reductions ofunsaturated compounds of commercial interest, and especially for thepreparation of chiral molecules, as taught by Simon et al. (Ang. Chem.Int. Ed. Engl. 1985, 24, 539-53). Unfortunately, most enzymes capable ofcatalyzing such reduction reactions require the presence of cofactors,which function as biological reducing agents. One broad class of enzymescapable of selective reduction and/or hydrogenation of unsaturatedorganic compounds are the nicotinamide dependent oxidoreductases, asdiscussed by Walsh (Enzymatic Reaction Mechanisms Freeman & Co., N.Y.,1979; pages 311-521).

[0005] Nicotinamide dependent oxidoreductases require the presence ofnicotinamide cofactors. The structures of the naturally occurringnicotinamide cofactors, (NAD⁺, NADP⁺, NADH and NADPH) are shown below.

[0006] A reduced nicotinamide cofactor (NADH or NADPH) binds to thenicotinamide cofactor dependent enzyme, and transfers a “hydride” (twoelectrons and one hydrogen nucleus) to reduce a substrate that alsobinds to the enzyme. After the substrate is reduced, the enzyme releasesthe oxidized form of the nicotinamide cofactor (NAD⁺ or NADP⁺).Biological systems typically recycle the oxidized nicotinamidecofactors, by employing an external reducing agent, in combination withother enyzmes, to regenerate the reduced form of the nicotinamidecofactors. In these nicotinamide cofactors the nicotinamide ring (shownschematically immediately below) is the reactive group. To regeneratethe reduced cofactor, an external reducing agent must transfer theequivalent of a “hydride” to the oxidized (pyridinium) form of thecofactor, regioselectively to form the reduced (1,4-dihydropyridine)form of the cofactor.

[0007] Although nicotinamide cofactor dependent enzymes and nicotinamidecofactors are present in all living organisms at low concentrations,they tend to be chemically unstable under non-biological conditions, andare extremely expensive in purified form. Because of their high cost,most industrial processes that seek to employ a combination of enzymesand nicotinamide cofactors must supply a method to regenerate thenicotinamide cofactors.

[0008] A number of methods for cofactor regeneration are known, asdiscussed by Chenault and Whitesides (Appl. Biochem. Biotechnol. 1987,14, 147-97), and in Enzymes in Organic Synthesis K. Drauz, H. Waldeman,Eds.; VCH: Weinheim, 1995; pages. 596-665. The most widely used methodsfor cofactor regeneration employ a chemical reducing agent and secondenzyme to regenerate the nicotinamide cofactors. For example, usingglucose as a reducing agent, glucose oxidase has been shown tosuccessfully regenerate NADP⁺/NADPH through up to 4×10⁴ turnovers (seeWong, and Whitesides J. Org. Chem. 1982, 47, 2816-18; Wong et al., J.Am. Chem. Soc. 1985, 107, 4028-31; Obon et al., Biotech. Bioeng. 1998,57, 510-17). Hummel et al., (Appl. Microbiol. Biotechnol. 1987, 26,409-416) have shown that a combination of formate dehydrogenase andformate salts regenerates NADH from NAD⁺ with turnover numbers for thereduced cofactor as high as 6×10⁵. In these methods, a second enzymecouples the regenerated NADH to substrate reduction. In cases whereactivity of two separate enzyme systems can be accomplished in vitrowithout undue complexity or expense, reduction of substrates with achemical reducing agent and two enzymes can be a viable cofactorregeneration method.

[0009] Prior art attempts to electrochemically regenerate thenicotinamide cofactors avoid the need for a second enzyme, but directelectrochemical methods have typically not achieved adequate cofactorregeneration, primarily due to formation of inactivenicotinamide-dimers. The addition of certain types of electron transfercatalysts or “mediators” to electrochemical methods can greatly improveelectrochemical regeneration, as disclosed by Steckhan (Topics inCurrent Chemistry, 1994, 170, 83-111). The most successful mediators arethe rhodium complexes disclosed by Steckhan et al (Ang. Chem,. 1982, 94,786; U.S. Pat. No. 4,526,661 and Organometallics 1991 10, 1568-77).Although these electrochemically-based systems have been successfullycoupled to enzymatic reduction reactions, thus far cofactor turnovernumbers remain too low to be commercially viable.

[0010] Photochemically assisted methods for chemical reduction ofNAD(P)⁺ to NAD(P)H in the presence of similar rhodium electron transfercatalysts, and successful coupling to enzymes has been reported(Willner, et al., in J. Am. Chem. Soc., 1984, 106, 5352-53, and J. Chem.Soc., Perkin Trans., 2 1990, 559-64; Franke and Steckhan in Angew. Chem.Intl. Ed. Engl., 1988, 27, 265; and Aono and Okura in Inorg. Chim. Acta,1988, 152, 55-59). Nevertheless, an economically competitive andlong-lived photo-chemical cofactor regeneration system which achievescofactor regeneration at rates and efficiencies competitive withenzymatic methods has remained an elusive goal.

[0011] Cofactor regeneration with non-biological chemical reducingagents is a simple approach, but most chemical reducing agents are notdesirably selective for production of 1,4-dihydro isomers of thecofactor nicotinamide ring, as discussed by Ohnishi and Tanimoto(Tetrahedron Lett. 1977, 1909-12). Dithionite salts are preferredreducing agents in this regard, providing up to about 10² turnovers ofthe nicotinamide cofactor, as described by Jones, et al. (J. Chem. Soc.,Chem. Commun., 1972, 856-57). Nevertheless, dithionite salts areincompatible with many enzymes and react directly with many substrates,are expensive, and generate undesirable sulfur-containing wastes.Steckhan reported the use of formate salts to directly reduce PEG-NAD⁺in a membrane reactor, in the presence of homogeneous rhodium catalystshaving covalently bound polyethyleneglycol tails (Angew. Chem., 1990,102, 445-7). Keinan, et al. (J. Am. Chem. Soc. 1986, 108, 162-9)reported the use of hydride donor alcohols (such as isopropanol) and analcohol dehydrogenase from T. brockii, in a “coupled substrate” methodto reduce certain organic substrates. In the “coupled substrate” methodone enzyme catalyzes both (a) reduction of NADP⁺ to NADPH by the hydridedonor alcohol, and (b) reduction of ketone substrates such as2-heptanone by NADPH.

[0012] Dihydrogen (H₂), is a highly desirable chemical reducing agent.H₂ is a strong reducing agent, and can be inexpensively produced andstored in high purity on a large scale. H₂ is typically innocuoustowards enzymes and cofactors, and because it is completely consumed inmost reduction reactions, it leaves no residues to complicatepurification or create chemical waste. Many examples are known of theuse of H₂ as a reducing agent in the presence of transition metalcatalysts (in the absence of enzymes or cofactors).

[0013] Nevertheless, the use of hydrogen as a reducing agent inconjunction with enzymes has only been possible with complex multi-stepor multi-component catalyst systems that employ indirect coupling of theof H₂ to cofactor regeneration. Wong et al (J. Am. Chem. Soc. 1981, 103,6227-8), and Otsuka, et al. (J. Mol. Catal. 1989, 51, 35-9), havereported the use H₂ and hydrogenase enzymes, which are air-sensitive andnot readily available, to reduce electron receptors such asmethylviologen to produce radicals. The radicals can be coupled via asecond enzyme (ferredoxin reductase) to NADH regeneration. The NADH iscoupled to substrate reduction via a third enzyme.

[0014] Abril and Whitesides (J. Am. Chem. Soc. 1982, 104, 1552-54)reported a multi-component approach in which a water soluble rhodiumcomplex of a bidentate phosphine ligand was employed to activate H₂, buttwo other enzymes and high concentrations of a lactate/pyruvate hydrogencarrier intermediate were required for substrate reduction.

[0015] A recent report by Bhaduri, et al. (J. Am. Chem. Soc. 1998, 120,12127-28) describes the use of H₂ to reduce NAD⁺ to NADH via a“secondary coupling” system. H₂ reacts with a platinum carbonyl clusterin a methylene chloride phase and reduces a redox-active dye (SafranineO). The reduced dye then diffuses to an aqueous phase where it reducesNAD⁺ to produce NADH. The NADH then combines with lactate dehydrogenaseto reduce pyruvic acid to lactic acid. Nevertheless, the platinumcarbonyl clusters are insoluble and unstable in water, necessitating theuse of the redox-active dye and a two phase solvent system. No enzymesother than lactate dehydrogenase, or substrates other than pyruvic acidwere reported.

[0016] Despite the potential advantages of the use of H₂ as a reducingagent for regenerating nicotinamide cofactors, simple, effective, andeconomically attractive methods for doing so have not been achieved.There is an unmet need in the art for simple and effective methods forcombining the low cost and environmental desirability of H₂ as areducing agent with the exquisite selectivity of enzyme catalysis. It isto such a desirable object that the present invention is primarilydirected.

SUMMARY OF THE INVENTION

[0017] The present invention meets the unmet needs in the art, byproviding processes and catalyst compositions, which employ H₂ as areducing agent for unsaturated organic compounds in the presence ofcatalysts comprising enzymes.

[0018] In accordance with the purpose(s) of this invention, as embodiedand broadly described herein, the invention therefore relates to, in oneaspect, a process for reducing an unsaturated organic compound,comprising mixing the unsaturated organic compound and H₂ in thepresence of a catalyst to form a reduced organic product, wherein thecatalyst comprises:

[0019] a) at least one metal salt or complex,

[0020] b) at least one nicotinamide cofactor; and

[0021] c) a nicotinamide cofactor dependent enzyme,

[0022] wherein:

[0023] i) when the metal salt or complex is a platinum carbonyl clustercomplex, the catalyst does not comprise a redox active dye; and

[0024] ii) when the metal salt or complex is a rhodium phosphinecomplex, the nicotinamide cofactor dependent enzyme is not a mixture ofhorse liver alcohol dehydrogenase and lactate dehydrogenase.

[0025] In another aspect, the invention provides a process for reducingan unsaturated organic compound, comprising mixing the unsaturatedorganic compound and H₂ in the presence of a catalyst to form a reducedorganic product, wherein the catalyst comprises:

[0026] a) a substantially aqueous buffer solution having a pH from about6.5 to about 9.0,

[0027] b) a water-soluble metal salt or complex comprising iron,ruthenium, osmium, cobalt, rhodium, iridium, nickel, palladium, orcopper,

[0028] c) a nicotinamide cofactor comprising NAD⁺, NADH, NADP⁺, NADPH,or a mixture thereof; and

[0029] d) one nicotinamide cofactor dependent enzyme classified underthe EC system as an 1.x.1.y. class enzyme, wherein x is 1,3,4, 5 or 10.

[0030] In a different aspect, the invention provides a process forreducing an unsaturated organic compound, comprising:

[0031] a) contacting H₂ and a catalyst, and

[0032] b) contacting an unsaturated organic compound with the catalystto form a reduced organic product,

[0033] wherein the catalyst comprises:

[0034] i) at least one metal salt or complex,

[0035] ii) at least one nicotinamide cofactor; and

[0036] iii) a nicotinamide cofactor dependent enzyme,

[0037] and wherein

[0038] iv) when the metal salt or complex is a platinum carbonyl clustercomplex, the catalyst does not comprise a redox active dye; and

[0039] v) when the metal salt or complex is a rhodium phosphine complex,the nicotinamide cofactor dependent enzyme is not a mixture of horseliver alcohol dehydrogenase and lactate dehydrogenase.

[0040] In yet another aspect, the invention provides a process forreducing an unsaturated organic compound, comprising:

[0041] a) contacting H₂, at least one metal salt or complex, and atleast one nicotinamide cofactor to form at least some reducednicotinamide cofactor, and

[0042] b) contacting the reduced nicotinamide cofactor, a nicotinamidecofactor dependent enzyme, and an unsaturated organic compound underconditions effective to form at least some of a reduced organic product,

[0043] wherein

[0044] i) when the metal salt or complex is a platinum carbonyl clustercomplex, the catalyst does not comprise a redox active dye; and

[0045] ii) when the metal salt or complex is a rhodium phosphinecomplex, the nicotinamide cofactor dependent enzyme is not a mixture ofhorse liver alcohol dehydrogenase and lactate dehydrogenase.

[0046] The invention further relates to a process comprising addingdihydrogen, H₂, to an unsaturated organic substrate using as a catalysta mixture comprising an enzyme, a nicotinamide cofactor, a metal salt orcomplex and optionally ligands, wherein the metal of the metal salt orcomplex is selected from iron, cobalt, nickel, copper, ruthenium,palladium, osmium, and iridium.

[0047] The invention also relates to a composition for reducingunsaturated organic compounds comprising H₂ and a catalyst, the catalystcomprising:

[0048] a) at least one metal salt or complex,

[0049] b) at least one nicotinamide cofactor; and

[0050] c) a nicotinamide cofactor dependent enzyme,

[0051] wherein

[0052] i) when the metal salt or complex is a platinum carbonyl clustercomplex, the catalyst does not comprise a redox active dye; and

[0053] ii) when the metal salt or complex is a rhodium phosphinecomplex, the nicotinamide cofactor dependent enzyme is not a mixture ofhorse liver alcohol dehydrogenase and lactate dehydrogenase.

[0054] In another embodiment, the invention relates to a process forstabilizing the activity of an oxidoreductase enzyme, comprising mixinga phosphine or phosphite with an oxidoreductase enzyme.

[0055] Additional advantages of the invention will be set forth in partin the description that follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] The present invention may be understood more readily by referenceto the following detailed description of preferred embodiments of theinvention and the Examples included therein and to the Figures and theirprevious and following description. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

[0057] It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an aromatic compound” includes mixtures ofaromatic compounds.

[0058] Often, ranges are expressed herein as from “about” one particularvalue, and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

[0059] In this specification and in the claims that follow, referencewill be made to a number of terms that shall be defined to have thefollowing meanings:

[0060] A weight percent of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

[0061] A residue of a chemical species, as used in the specification andconcluding claims, refers to the moiety that is the resulting product ofthe chemical species in a particular reaction scheme or subsequentformulation or chemical product, regardless of whether the moiety isactually obtained from the chemical species. Thus, an ethylene glycolresidue in a polyester refers to one or more —OCH₂CH₂O— repeat units inthe polyester, regardless of whether ethylene glycol is used to preparethe polyester. Similarly, a sebacic acid residue in a polyester refersto one or more —CO(CH₂)₈CO— moieties in the polyester, regardless ofwhether the residue is obtained by reacting sebacic acid or an esterthereof to obtain the polyester.

[0062] “Optional” or “optionally” means that the subsequently describedevent or circumstance may or may not occur, and that the descriptionincludes instances where said event or circumstance occurs and instanceswhere it does not. For example, the phrase “optionally substituted loweralkyl” means that the lower alkyl group may or may not be substitutedand that the description includes both unsubstituted lower alkyl andlower alkyl where there is substitution.

[0063] By the term “effective amount” of a compound or property asprovided herein is meant such amount as is capable of performing thefunction of the compound or property for which an effective amount isexpressed. As will be pointed out below, the exact amount required willvary from process to process, depending on recognized variables such asthe compounds employed and the processing conditions observed. Thus, itis not possible to specify an exact “effective amount.” However, anappropriate effective amount may be determined by one of ordinary skillin the art using only routine experimentation.

[0064] In one aspect, the invention provides a process for reducing anunsaturated organic compound, comprising mixing the unsaturated organiccompound and H₂ in the presence of a catalyst to form a reduced organicproduct, wherein the catalyst comprises:

[0065] a) at least one metal salt or complex,

[0066] b) at least one nicotinamide cofactor; and

[0067] c) a nicotinamide cofactor dependent enzyme.

[0068] In additional embodiments of the invention, one or more of thefollowing further exclusions and/or limitations to the scope may apply:

[0069] a) the catalyst comprises one and only one enzyme,

[0070] b) the metal salt or complex does not comprise a platinumcarbonyl cluster complex,

[0071] c) the metal salt or complex does not comprise a rhodiumphosphine complex, and/or

[0072] d) the formation of the reduced organic product is capable ofoccurring in the absence of electrochemical or photochemical sources ofexternal energy.

[0073] It is intended that when it is stated that the catalyst comprises“one” enzyme, one, and only one, enzyme is present in the catalyst.

[0074] Platinum carbonyl cluster complexes are metal salts or complexeshaving three or more platinum atoms, wherein each platinum atom isbonded to at least one carbon monoxide ligand. Examples of commonplatinum carbonyl complexes include but are not limited to[Pt₉(CO)₁₈]²⁻, and [Pt₁₂(CO)₂₄]²⁻.

[0075] When the metal salt or complex is a platinum carbonyl clustercomplex; one or more of the following further exclusions and/orlimitations may apply:

[0076] a) the catalyst does not comprise a redox active dye, redoxactive dyes including but not being limited to Safranine O, methylviologen, methylene blue, or the like,

[0077] b) the unsaturated organic substrate is not a pyruvate,

[0078] c) the reduced organic product is not a lactate,

[0079] d) the enzyme does not comprise lactate dehydrogenase, or

[0080] e) the process does not comprise water-immiscible solvents, whichinclude but are not limited to methylene chloride, chloroform, and thelike.

[0081] Rhodium phosphine complexes are metal salts or complexes having arhodium atom bonded to a phosphine residue, the phosphine residue havingthe formula PR₃, wherein the three R groups independently comprisehydrocarbyl groups or residues. In other embodiments, the metal salt orcomplex does not comprise a rhodium bis(phosphine) complex. Rhodiumbis(phosphine) complexes have a rhodium atom bonded to two phosphineresidues, that may or may not be bonded to each other through a bridginggroup, to form a bidentate phosphine ligand. In some embodiments, themetal salts or complexes do not comprise a water soluble rhodiumphosphine complex.

[0082] When the metal salt or complex is a rhodium phosphine complex,one or more of the following further exclusions and/or limitations tothe scope of certain embodiments of the present invention may apply:

[0083] a) the nicotinamide cofactor dependent enzyme is not lactatedehydrogenase,

[0084] b) the nicotinamide cofactor dependent enzyme is not horse liveralcohol dehydrogenase,

[0085] c) the process does not comprise lactate or pyruvate, and/or

[0086] d) the unsaturated organic substrate does not comprisecyclohexanone or 2-norbornanone.

[0087] In preferred embodiments of the processes of the currentinvention, the formation of the reduced organic product is capable ofoccurring in the absence of electrochemical or photochemical sources ofexternal energy. In other words, the preferred processes of the presentinvention only require thermal energy to successfully reduce unsaturatedorganic compounds with H₂, and they do not require the presence orsupply of visible or ultraviolet light, or the presence or supply ofsurfaces or electrodes supplied with electromotive force differentialsin order to induce the formation of reduced organic products. Moreparticularly, preferred embodiments of the present invention do notrequire the presence or supply of light, or the presence or supply ofsurfaces or electrodes supplied with electromotive force differentialsto initiate the reaction of H₂ with the metal salt or complex componentof the catalyst.

[0088] The order of mixing of the H₂, the unsaturated organic compound,the metal salt or complex, the nicotinamide cofactor, and thenicotinamide cofactor dependent enzyme may occur in any order, orsimultaneously.

[0089] In a preferred embodiment, the invention provides a process forreducing an unsaturated organic compound, comprising mixing theunsaturated organic compound and H₂ in the presence of a catalyst toform a reduced organic product, wherein the catalyst comprises:

[0090] a) at least one metal salt or complex,

[0091] b) at least one nicotinamide cofactor; and

[0092] c) a nicotinamide cofactor dependent enzyme,

[0093] wherein:

[0094] i) when the metal salt or complex is a platinum carbonyl clustercomplex, the catalyst does not comprise a redox active dye; and

[0095] ii) when the metal salt or complex is a rhodium phosphinecomplex, the nicotinamide cofactor dependent enzyme is not a mixture ofhorse liver alcohol dehydrogenase and lactate dehydrogenase.

[0096] The unsaturated organic compounds of the invention are carboncontaining molecules that have at least one multiple bond between twoatoms of the compound, X and Y. The atoms X and Y may be any atom of thePeriodic Table capable of forming multiple bonds. Preferably, the X andY atoms are independently selected from carbon, nitrogen, oxygen,sulfur, and phosphorus atoms. Preferably, at least one of the X or Yatoms is a carbon atom. In many preferred embodiments, one of the X or Yatoms is a carbon atom, and the other atom is carbon, oxygen or nitrogenatom. Preferably, the multiple bond between the atoms X and Y is adouble bond, or a triple bond. The unsaturated organic compounds canhave more than one multiple bond, and the multiple bonds may formconjugated combinations of multiple bonds.

[0097] Examples of classes of unsaturated organic compounds of theinvention include but are not limited to

[0098] a) a ketone, an aldehyde, a carboxylic acid, a carboxylic acidester, or an amide,

[0099] b) an α-β unsaturated derivative of a ketone, an aldehyde, acarboxylic acid, a carboxylic acid ester, or an amide,

[0100] c) a fatty acid, a monoglyceride, a diglyceride, or atriglyceride, having an olefinic unsaturated group,

[0101] d) an olefin, an aromatic compound or a heteroaromatic compound,

[0102] e) an imine, or an oxime,

[0103] f) a sugar, an amino acid, a peptide, or a protein, wherein thesugar, amino acid, peptide, or protein has an unsaturated group.

[0104] Examples of unsaturated organic compounds include but are notlimited to acetoin, 1,1-dimethoxyacetone, glycerone, acetophenone,2-acetylfuran, hydroxy-acetone, 6-methyl-5-heptene-2-one,5-norbornene-2-one, 2-heptanone, 8-oxo-2-nonanone, cyclopropyl methylketone, L-sorbose, 2,4,6/3,5-pentahydroxycyclohex-anone, aldose,glyoxylate, pyruvate, acetoacetate, ethyl 4-chloroacetoacetate, ethyl4-oxo-hexanoate, oxaloacetate, 2,5-diketo-D-gluconic acid,D-glucono-1,5-lactone, 5α-androstane-3,17-dione,androst-4-ene-3,17-dione, L lactaldehyde, fructose, D-glyceraldehyde,orotate, 2-oxoglutarate, 3-hydroxypyruvate, glyoxylate, and L-lysine.

[0105] The reduced organic products of the invention typicallycorrespond in structure to the starting unsaturated organic compoundreduced, or a residue thereof, in which the multiple bond between the Xand Y atoms is cleaved in a reduction process. Reduction is broadlydefined for the purposes of this disclosure as the addition of one ormore electrons to the multiple bond, the electrons having been donatedby another compound, termed a reducing agent. Rearrangement and/orcleavage of functional groups may also occur during or subsequent to thereduction of the multiple bond. Often, reduction of the multiple bond isaccompanied by the bonding of one or more hydrogen atoms to at least oneof the X and Y atoms.

[0106] In preferred embodiments, the unsaturated organic compoundcomprises a carbon atom doubly bonded to another carbon, oxygen, ornitrogen atom, the reducing agent is H₂, and the reduced organiccompound has a structure corresponding to the addition of two hydrogenatoms to the double bond, as illustrated below, in which case thereduction reaction may also be termed a hydrogenation reaction.

[0107] If the S1, S2 groups and/or Y groups illustrated in the equationabove are different, reduction and formation of the new carbon-hydrogenbond can result in the formation of two enantiomeric isomers of thereduced organic product. The catalysts of the invention, which compriseenzymes, often selectively produce only one of the two possibleenantiomeric isomers.

[0108] The processes of the invention occur in the presence of acatalyst comprising at least the following components:

[0109] a) at least one metal salt or complex,

[0110] b) at least one nicotinamide cofactor; and

[0111] c) a nicotinamide cofactor dependent enzyme.

[0112] The components of the catalyst need not, but often do comprise asingle phase or mixture, so long as the component can interact in asuitable manner. The components of the catalysts can be dissolved in oneor more solvents, or can be dispersed upon or bonded to one or moresupport phases. In preferred embodiments of the invention, the threecomponents of the catalyst comprise a mixture that is dispersed orsubstantially dissolved in a liquid medium. Preferably, the liquidmedium comprises a homogeneous liquid phase. The liquid medium maycomprise an organic solvent, water, or a mixture thereof. Preferably,the liquid medium comprises a substantially aqueous phase. In manypreferred embodiments, the liquid medium comprises an aqueous buffersolution having a pH from about 6.5 to about 9.0, which typicallymaximizes the stability of the nicotinamde cofactor dependent enzymesand the nicotinamide cofactors. More preferably, the pH of the aqueousbuffer solution is between about 7.0 to about 8.5. Aqueous buffersolutions comprising phosphate salts are preferred aqueous buffersolutions.

[0113] The nicotinamide cofactors employed in the invention include thenaturally occurring nicotinamide adenine dinucleotides describedhereinabove, and further include any structural analogs thereof that arecapable of effectively interacting with the nicotinamide cofactordependent enzymes of the invention to reduce unsaturated organiccompounds. Structural analogs of the naturally occurring cofactorscomprise compounds wherein the naturally occurring structure is modifiedby the addition of or removal of one or more functional groups.Preferably, the nicotinamide cofactors of the invention are thebiologically preferred cofactors NAD⁺, NADH, NADP⁺, NADPH, or a mixturethereof. It is well known many nicotinamide cofactor dependent enzymewill preferentially bind and/or utilize only specifically phosphorylatedcofactors (such as NADP⁺, and NADPH), while other nicotinamide cofactordependent enzyme will preferentially bind and/or utilize only bindand/or utilize non-phosphorylated cofactors (such as NAD⁺, NADH).

[0114] The nicotinamide cofactor dependent enzymes employed in theinvention comprise any naturally occurring or biotechnologicallymodified or engineered enzyme that requires the presence of anicotinamide cofactor, or an analog thereof, in order to reduce oroxidize organic substrates. It is to be understood that while manynicotinamide cofactor dependent enzymes catalyze oxidation reactions innature, they are nevertheless often useful for catalyzing the reductionprocesses of the invention.

[0115] The Enzyme Commission of International Union of Biochemistry andMolecular Biology (“EC”) has devised a well-known four digit numericalsystem for classifying enzymes, in terms of the type of reaction thatthe enzymes catalyze. The EC classification system has been described byDixon, Webb, Thorne, and Tipton (“Enzymes”, Chapter 5, pages 207-230,Academic Press, 1979), which is hereby incorporated by reference in itsentirety, for the purposes of describing the classification of enzymesand the relationship of the classifications to the substrates andproducts of the reactions catalyzed by the enzymes.

[0116] The first digit of an Enzyme Classification (“EC”) corresponds toone of six classes of enzymes. The nicotinamide cofactor dependentenzymes of the present invention are all members of the class ofoxidoreductases, which comprise enzymes that mediate the transfer ofelectrons, H atoms, or hydride atoms. Oxidoreductases all have a firstdigit EC classification of “1”.

[0117] The second digit of an EC classification relates to subclasses ofthe enzymes specified by the first digit. For an oxidoreductase, thesecond digit pertains to a group of twenty subclasses of functionalgroups of the substrates and/or products for the enzyme, i.e. classes ofthe functional groups in the substrates or products which undergooxidation or reduction. The third digit of an EC classification relatesto another series of sub-subclasses. In the case of the oxidoreductases,a third digit of “1” indicates an oxidoreductase that requires thepresence of nicotinamide cofactors. Therefore, the nicotinamide cofactordependent enzymes of the present invention are all classified under theEC system as “1.x.1.y.” class enzymes. The fourth digit of and ECclassification specifies a serial number for the enzyme, which is oftenrelated to the particular identity of the natural substrate of theenzyme.

[0118] Preferred embodiments of the invention employ nicotinamidecofactor dependent enzymes, which may be further defined by the valuesof x and y in the EC classification of the enzyme. The second digit ofan EC classification of an enzyme, i.e., the value of x, corresponds tothe class of functional group oxidized or reduced by the enzyme.Preferred enzymes of the invention have x values of 1, 3, 4, 5, or 10,corresponding to ketone or aldehyde reductions (x=1); olefin reduction(x=3); imine reductions (x=4 or 5); and reduction of diphenols orascorbate (x=10). Therefore in preferred embodiments of the presentinvention, the nicotinamide cofactor dependent enzyme is an enzymeclassified under the EC system as an 1.x.1.y. class enzyme, wherein x is1, 3, 4, 5 or 10. More than 340 enzymes are presently known to fallwithin those preferred classes of enzymes.

[0119] For enzymes within the class of 1.1.1 .y enzymes, Table 1 belowrelates the value of y to the name of the enzyme and its naturalsubstrates. TABLE 1 1.1.1.y Class Enzymes. y Name of Enzyme 1 alcoholdehydrogenase 2 alcohol dehydrogenase 4 butanediol dehydrogenase 5diacetyl reductase 6 glycerol dehydrogenase 7 glyerol-3-phosphatedehydrogenase 14 L-iditol 2-dehydrogenase 18 myo-inositol2-dehydrogenase 21 aldose reductase 22 UDP glucose 6-dehydrogenase 26glyoxalate dehydrogenase 27 L-lactate dehydrogenase 30 3-hydroxybutyratedehydrogenase 37 malate dehydrogenase 40 malate dehydrogenase 41isocitrate dehydrogenase 42 isocitrate dehydrogenase 44 phosphogluconatedehydrogenase 47 glucose 1-dehydrogenase 48 galactose 1-dehydrogenase 49glucose-6-phosphate 1-dehydrogenase 50 3a-hydroxysteroid dehydrogenase51 3 (or 17) b-hydroxysteroid dehydrogenase 53 3a (or 20b)hydroxysteroid dehydrogenase 55 lactaldehyde reductase 67 mannitol2-dehydrogenase 72 glycerol dehydrogenase 83 D-malate dehydrogenase 95glycerol dehydrogenase 119 glucose 1-dehydrogenase 122 D-threo-aldose1-dehydrogenase 159 12a-hydroxysteroid dehydrogenase 17612a-hydroxysteroid dehydrogenase

[0120] For enzymes within the class of 1.3.1 .y enzymes, enzymes ofclass 1.3.1.14 i.e. orotate reductases are preferred. (NADH)

[0121] For enzymes within the class of 1.4.1 .y enzymes, Table 2 belowrelates the value of y to the name of the enzyme and/or it's naturalsubstrate. TABLE 2 1.4.1.y Class Enzymes. y Name of Enzyme 1 alaninedehydrogenase 2 glutamate dehydrogenase 3 glutamate dehydrogenase 4glutamate dehydrogenase 5 L-amino-acid dehydrogenase 7 serinedehydrogenase 8 valine dehydrogenase 9 leucine dehydrogenase 10 glycinedehydrogenase 11 L-erythro-3,5-diaminohexanoate dehydrogenase 122,4-diaminopentanoate dehydrogenase 13 glutamate synthase 14 glutamatesynthase 15 lysine dehydrogenase 16 diaminopimelate dehydrogenase 17N-methylalanine dehydrogenase 18 lysine 6-dehydrogenase 19 tryptophanedehydrogenase 20 phenylalanine dehydrogenase

[0122] In preferred embodiments of the invention, the nicotinamidecofactor dependent enzyme is an enzyme classified under the EC system asa 1.1.1.1, a 1.1.1.2, a 1.1.1.4, a 1.1.1.5, a 1.1.1.6, a 1.1.1.7, a1.1.1.14, a 1.1.1.18, a 1.1.1.21, a 1.1.1.22, a 1.1.1.26, a 1.1.1.27, a1.1.1.30, a 1.1.1.37, a 1.1.1.40, a 1.1.1.41, a 1.1.1.42, a 1.1.1.44, a1.1.1.47, a 1.1.1.48, a 1.1.1.49, a 1.1.1.50, a 1.1.1.51, a 1.1.1.53, a1.1.1.55, a 1.1.1.67, a 1.1.1.72, a 1.1.1.83, a 1.1.1.95, a 1.1.1.119, a1.1.1.122, a 1.1.1.159, a 1.1.1.176, a 1.3.1.14, a 1.4.1.1, a 1.4.1.2, a1.4.1.3, a 1.4.1.4, a 1.4.1.5, a 1.4.1.7, a 1.4.1.8, a 1.4.1.9, a1.4.1.10, a 1.4.1.11, a 1.4.1.12, a 1.4.1.13, a 1.4.1.14, a 1.4.1.15, a1.4.1.16, a 1.4.1.17, a 1.4.1.18, a 1.4.1.19 or a 1.4.1.20 class enzyme.

[0123] In more preferred embodiments of the invention, the nicotinamidecofactor dependent enzyme is an enzyme classified under the EC system as1.1.1.1, 1.1.1.2, 1.1.1.5, 1.1.1.6, 1.1.1.7, 1.1.1.14, 1.1.1.18,1.1.1.21, 1.1.1.26, 1.1.1.27, 1.1.1.37, 1.1.1.40, 1.1.1.41, 1.1.1.42,1.1.1.47, 1.1.1.48, 1.1.1.49, 1.1.1.72, 1.1.1.83, 1.1.1.95, 1.1.1.119,14, 1.4.1.1, 1.4.1.3, 1.4.1.4, 1.4.1.9 or 1.4.1.20 class enzyme.

[0124] In other preferred embodiments of the invention the nicotinamidecofactor dependent enzyme is an enzyme classified under the EC system asa 1.1.1.1 or 1.1.1.2, class enzyme.

[0125] In the present invention, the specification of an enzyme by itsEC classification does not necessarily strictly limit the classes orspecies of unsaturated organic compounds that may be reduced by theenzyme in the processes of the invention, or the reduced organicproducts produced. In many embodiments of the present invention, themethods of the invention will reduce unnatural unsaturated organiccompounds, to produce unnatural reduced organic products, which will notbe literally specified by the EC classification of an enzyme.

[0126] Nevertheless, it is to be understood that the 2^(nd) and/or4^(th) digits of the EC classification of a particular oxidoreductaseenzyme inherently identifies preferred classes of oxidation and/orreduction reactions that may be catalyzed by the enzyme, and/or thecorresponding classes and/or species of substrates and products that maybe involved. A listing of the names, EC classifications and thecorresponding chemical reactions of the oxidoreductase enzymes relevantto the present invention may found in “Enzyme Nomenclature1992—Recommendations of the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology on the Nomenclature andClassification of Enzymes”, pages 1-154, Academic Press, 1992, which ishereby incorporated by reference, for the purposes of describing theclassification of enzymes and the relationship of the classifications tothe substrates and products of the reactions catalyzed by the enzymes.Of particular relevance are pages 24-55 (1.1.1.y class enzymes), pages65-67 (1.2.1.y class enzymes), pages 76-83 (1.3.1.y class enzymes),pages 87-89 (1.4.1.y class enzymes), pages 93-96 (1.5.1.y classenzymes), and 1.13 (1.10.1.y class enzymes).

[0127] For example, if the nicotinamide cofactor dependent enzyme is analcohol dehydrogenase [1.1.1.1] or [1.1.1.2], a preferred reducedorganic product is an alcohol.

[0128] In a similar manner, preferred classes or species of reducedorganic products are hereinbelow identified for a number of particularEC enzyme classifications. For example, if the nicotinamide cofactordependent enzyme is an aldose reductase [1.1.1.21], a preferred reducedorganic product is an alditol. If the nicotinamide cofactor dependentenzyme is a glyoxolate reductase [1.1.1.26], a preferred reduced organicproduct is a glycolate or glycerate.

[0129] With respect to the identification of preferred species ofreduced organic products for particular enzymes: If the nicotinamidecofactor dependent enzyme is a diacetyl reductase [1.1.1.5], a preferredreduced organic product is 3-hydroxy-2-butanone (acetoin). If thenicotinamide cofactor dependent enzyme is a glycerol dehydrogenase[1.1.1.6], a preferred reduced organic product is glycerol. If thenicotinamide cofactor dependent enzyme is an propanediol-phosphatedehydrogenase [1.1.1.7], a preferred reduced organic product ispropane-1,2-diol 1-phosphate. If the nicotinamide cofactor dependentenzyme is an L-lactate dehydrogenase [1.1.1.27], a preferred reducedorganic product is an (S)-lactate. If the nicotinamide cofactordependent enzyme is a malate dehydrogenase [1.1.1.37], a preferredreduced organic product is (S)-malate. If the nicotinamide cofactordependent enzyme is malate dehydrogenase [1.1.1.40], a preferred reducedorganic product is (S)-malate. If the nicotinamide cofactor dependentenzyme is isocitrate dehydrogenase [1.1.1.41], a preferred reducedorganic product is isocitrate. If the nicotinamide cofactor dependentenzyme is isocitrate dehydrogenase [1.1.1.42], a preferred reducedorganic product is isocitrate.

[0130] Moreover, if the nicotinamide cofactor dependent enzyme isglucose 1-dehydrogenase [1.1.1.47], a preferred reduced organic productis β-D-glucose. If the nicotinamide cofactor dependent enzyme isgalactose 1-dehydrogenase [1.1.1.48], a preferred reduced organicproduct is D-galactose. If the nicotinamide cofactor dependent enzyme isglucose 6-phosphate 1-dehydrogenase [1.1.1.49], a preferred reducedorganic product is D-glucose 6-phosphate. If the nicotinamide cofactordependent enzyme is glycerol dehydrogenase (NADP) [1.1.1.72], apreferred reduced organic product is glycerol. If the nicotinamidecofactor dependent enzyme is D-malate dehydrogenase [1.1.1.83], apreferred reduced organic product is (R)-malate. If the nicotinamidecofactor dependent enzyme is phosphoglycerate dehydrogenase [1.1.1.95],a preferred reduced organic product is 3-phosphoglycerate. If thenicotinamide cofactor dependent enzyme is glucose 1-dehydrogenase (NADP)[1.1.1.119], a preferred reduced organic product is D-glucose,D-mannose, 2-deoxy-D-glucose or 2-amino-2-deoxy-D-mannose.

[0131] Additionally, if the nicotinamide cofactor dependent enzyme isalanine dehydrogenase [1.4.1.1], a preferred reduced organic product isL-alanine. If the nicotinamide cofactor dependent enzyme is glutamatedehydrogenase [1.4.1.3], a preferred reduced organic product isL-glutamate. If the nicotinamide cofactor dependent enzyme is glutamatedehydrogenase [1.4.1.4], a preferred reduced organic product isL-glutamate. If the nicotinamide cofactor dependent enzyme is leucinedehydrogenase [1.4.1.9], a preferred reduced organic product isL-leucine. If the nicotinamide cofactor dependent enzyme isphenylalanine dehydrogenase [1.4.1.20], a preferred reduced organicproduct is L-phenylalanine.

[0132] It is to be understood that in the preceding description relatingthe EC classifications of preferred enzymes to their substrates, theinvention further provides methods for reducing man-made structuralanalogs of the natural substrates, to produce unnatural reduced organicproducts

[0133] It has been found that the maintenance of optimal activity of theenzymes is improved in the presence of certain stabilizers. Therefore,in preferred embodiments, the processes and compositions of theinvention further comprise a stabilizer for the nicotinamide cofactordependent enzyme. Preferably the stabilizers comprise sulfur orphosphorus compounds. Preferred sulfur containing stabilizers havesulfhydril groups, and include compounds exemplified by dithiothreitol(DTT), mercaptoethanol, and the like. Preferred phosphorus containingstabilizers have phosphorus atoms with oxidizable pairs of unsharedelectrons, such as phosphines, or phosphites, which include compoundssuch as tris(m-sulfonatophenyl)phosphine trisodium salt (TPPTS), or1,3,5-triaza-7-phosphaadamantane (PTA).

[0134] Most of the nicotinamide cofactor dependent enzymes of theinvention have cysteine amino acid residues. Nevertheless, preferrednicotinamide cofactor dependent enzymes of the invention do not comprisea cysteine amino acid residue, because such enzymes may not require thepresence of a stabilizer. Two cysteine-free enzymes which are preferredin the practice of the present invention are 2,5-diketo-D-gluconic acidreductase and glucose-6-phosphate and 1-dehydrogenase.

[0135] It is also to be understood that enzymes are often highlyselective for the production of only one of two possible enantiomers ofa reduced organic product. Therefore, in preferred embodiments, theinvention provides processes wherein the reduced organic product isproduced in a substantial enantiomeric excess.

[0136] The catalysts of the invention also comprise at least one metalsalt or complex. The metal salt or complex may comprise any compound,composition, or phase containing at least one transition metal element,lanthanide metal element, or actinide metal element from the PeriodicTable of the elements, with the proviso that the metal salt or complexis not nicotinamide cofactor dependent enzyme that contains a transitionmetal element, a lanthanide metal element, or an actinide metal elementas part of its structure. Preferably, the metal salt or complexcomprises a transition metal element selected from Groups 8, 9, 10, or11 of the Periodic Table. Preferred elements from Groups 8, 9, 10, or 11comprise, iron, ruthenium, osmium, cobalt, rhodium, iridium, nickel,palladium, platinum, or copper. In highly preferred embodiments of theinvention, the metal salt or complex comprises ruthenium, rhodium, orpalladium. The most preferred metal element is ruthenium.

[0137] The preferred oxidation state of the metal salt will vary withthe identity of the metal, process variables such as the medium, thepressure of H₂, and the identity of any ligands bonded to the metal. Itis to be understood that the oxidation state of the metal added to themixture may be altered by the presence of the other components of thereaction. Examples of species of metal salts or complexes include butare not limited to iron trichloride, ferrocene, iron pentacarbonyl,ruthenium trichloride hydrate, [Cl₂Ru(TPPTS)₂]₂, osmium tetraoxide,dicobalt octacarbonyl, rhodium trichlorided, [ClRh(P(C₆H₅)₃)₃],[ClRh(TPPTS)₃], nickel tetracarbonyl, nickel acetate,[Cl₂Pd(P(C₆H₅)₃)₂], Na₂[PtCl₆], CuBr₂, and the like.

[0138] In many embodiments of the invention, the metal salt or complexcomprises one or more ligands. Ligands are any organic or inorganiccompound that can coordinately, datively, or covalently bond to themetal atom of the metal salt or complex. Examples of suitable ligandsinclude but are not limited to water, a hydroxide, an oxide, an amine,an amide, an imine, an oxime, an imide, a nitrogen containingheterocycle, a nitrogen containing macrocycle, a nitrile, a phosphine, aphosphide, a phosphite, an alcohol, a thiol, an alcoholate, a thiolate,a sulfur containing heterocycle, an oxygen containing heterocycle, anether, a cyclic ether, a thioether; a phenol, a thiophenol, a phenolate,a thiophenolate; a halide, a hydride, a borohydride, a ketone, analdehyde, a carboxylic acid, an ester, an amino acid, a carboxylate, anacetonate, an iminate, an acetylacetonate, an iminoacetonate, animinoiminate, an alkene, an alkyne, a diene, an allyl residue, a dienylresidue a cyclopentadienyl residue, an indenyl residue, an arene, apolycyclic aromatic residue, a hydrocarbyl residue, carbon monoxide, acyanide, nitric oxide, H₂, substituted silyl residues, a sulfate, asulfoxide, a sulfone, a sulfonate, a phosphate, a phosphonate, or anyligand containing more than one of the above functional groups orresidues.

[0139] In preferred embodiments of the invention, the metal salt orcomplex has a ligand comprising a phosphine residue, a phosphiteresidue, carbon monoxide, a cyclopentadienyl residue, an aromaticresidue, a halide, or a hydride. A phosphine residue comprises atrivalent phosphorus residue having the formula PR₁R₂R₃. A phosphiteresidue comprises a trivalent phosphorus residue having at least onealkoxy residue in substitution for the R₁, R₂, or R₃ residues of aphosphine. Preferably, the R₁, R₂, and R₃ residues of the phosphinesand/or phosphites are independently selected from hydrogen, alkyl,alkylene, aryl, or halide residues.

[0140] In other preferred embodiments of the invention, the metal saltor complex has a ligand comprising a phosphine residue, a phosphiteresidue, a cyclopentadienyl residue, or an aromatic residue, wherein theligand has one or more polar functional groups. Preferred polarfunctional groups include one or more hydroxyl, carboxylic, amine,amide, ketone, aldehyde, nitro, and other similar polar substituentgroups for organic compounds. Preferred polar functional groups alsoinclude anionic groups, cationic groups, or poly(alkylene glycol)groups. Preferred anionic polar functional groups include carboxylates,sulfates, sulphonates, phosphates, phosphonates, and the like. Preferredcationic polar functional groups include ammonium groups, sulfoniumgroups, phosphonium groups, and the like. Preferred poly(alkyleneglycol) functional groups include poly(ethylene glycol) groups,polypropylene glycol groups, polybutylene glycol groups, and the like.

[0141] Although the polar functional groups may serve various purposes,such as modification of the properties and reactivity of the metalcomplex, in many preferred embodiments, the polar functional groupserves the purpose of increasing the water solubility of the ligandand/or the resulting metal salt or complex. A particularly preferredclass of ligands are water soluble phosphine or phosphite ligands, whichare useful as homogenous catalysts, as described by Kalck and Monteil(Adv. Organomet. Chem., 1992, 34, 219-284). A preferred class of watersoluble phosphine ligands comprises phosphine compounds having one ormore anionic sulfonate groups. A well known example of such compoundscomprises a salt of tris(m-sulfonatophenyl)phosphine. Another knownwater soluble phosphine ligand is 1,3,5-triaza-7-phosphaadamantane, asdescribed by. Daigle et al. (Inorg. Synth., 1998, 32, 40-45).

[0142] Preferred metal salts or complexes are significantly soluble inwater. Significant water solubility permits the metal salt or complex toreact with and/or activate H₂ in the water phase, and rapid transfer ofhydrogen to reduce the water soluble nicotinamide cofactors. Metal saltsor complexes are significantly soluble in water if the metal of themetal salt or complex is solubilized to the extent of at least about 1part per million in water. Preferably, the metal is soluble to theextent of greater than about 10 parts per million in water. Even morepreferably, the metal is soluble to the extent of greater than about 100parts per million in water.

[0143] H₂, i.e. dihydrogen, is the reducing agent supplied to theprocesses of the invention. H₂ has very significant practical andenvironmental advantages as compared with other chemical reducingagents, as discussed hereinabove. H₂ is also very economicallyattractive from a cost perspective. The table below illustrates therelative costs of a series of relevant reducing agents. TABLE 3 Cost ofOne Mole of Various “Hydride Reagents” Reducing Agent mol wt. $/mole H₂2     0.01^(a) glucose 180     0.24^(b) Na[CO₂H] 68     3^(b) NaBH₄ 38   32^(b) NADH 709  26,781^(c) NADPH 833 358,190^(c)

[0144] The H₂ may be present at any pressure which is effective toproduce at least some of the reduced organic product. The pressure of H₂that will produce at least some reduced organic product will vary withthe compositions of the catalyst, the reaction temperature, and otherreaction conditions, and variables. Preferably the H₂ is present at apressure less than about 100 atmospheres. More preferably, the H₂ ispresent at a pressure from about 0.1 atmospheres to about 50atmospheres. Even more preferably, the H₂ is present at a pressure fromabout 1 atmosphere to about 20 atmospheres.

[0145] The processes of the invention can occur at any temperature thatinduces the reduction of the unsaturated organic compounds, while notsubstantially denaturing the activity of the nicotinamide enzyme, orsubstantially deactivating the nicotinamide cofactors. Preferably, theprocesses of the invention are conducted at a temperature from about 0°C. to about 90° C. More preferably, the processes of the invention areconducted at a temperature from about 0° C. to about 45° C.

[0146] In a preferred embodiment, the invention provides a process forreducing an unsaturated organic compound, comprising mixing theunsaturated organic compound and H₂ in the presence of a catalyst toform a reduced organic product, wherein the catalyst comprises:

[0147] a) a substantially aqueous buffer solution having a pH from about6.5 to about 9.0,

[0148] b) a water-soluble metal salt or complex comprising iron,ruthenium, osmium, cobalt, rhodium, iridium, nickel, palladium, orcopper,

[0149] c) a nicotinamide cofactor comprising NAD⁺, NADH, NADP⁺, NADPH,or a mixture thereof; and

[0150] d) one nicotinamide cofactor dependent enzyme classified underthe EC system as an 1.x.1.y. class enzyme, wherein x is 1,3,4, 5 or 10.

[0151] In another preferred embodiment, the invention provides a processcomprising adding dihydrogen, H₂, to an unsaturated organic substrateusing as a catalyst a mixture comprising an enzyme, a nicotinamidecofactor, a metal salt or complex and optionally ligands, wherein themetal of the metal salt or complex is selected from iron, cobalt,nickel, copper, ruthenium, palladium, osmium, iridium. In even morepreferred embodiments, the metal of the metal salt or complex isselected from ruthenium and palladium.

[0152] It is clear that in the absence of a catalyst, the unsaturatedorganic compounds of the reaction typically do not react with H₂, andare not reduced by H₂ to form the reduced organic products of theinvention. Therefore, the invention provides a preferred process forreducing an unsaturated organic compound, comprising:

[0153] a) contacting H₂ and a catalyst, and

[0154] b) contacting an unsaturated organic compound with the catalystto form a reduced organic product,

[0155] wherein the catalyst comprises:

[0156] i) at least one metal salt or complex,

[0157] ii) at least one nicotinamide cofactor; and

[0158] iii) a nicotinamide cofactor dependent enzyme,

[0159] and wherein

[0160] iv) when the metal salt or complex is a platinum carbonyl clustercomplex, the catalyst does not comprise a redox active dye; and

[0161] v) when the metal salt or complex is a rhodium phosphine complex,the nicotinamide cofactor dependent enzyme is not a mixture of horseliver alcohol dehydrogenase and lactate dehydrogenase.

[0162] It is to be understood that in the embodiments of the inventiondescribed immediately above, H₂, the catalyst, and the unsaturatedorganic compound may all be present in a mixture or single phase, but itis not necessary that the unsaturated organic product is directlycontacted with H₂. In some embodiments, the catalyst (or certaincomponents thereof) might be contacted with H₂ in one reaction phase orreactor, while the unsaturated organic product was contacted withcatalyst in a different phase or reactor. The steps of contacting H₂ anda catalyst, and contacting an unsaturated organic compound with thecatalyst may occur simultaneously, or sequentially.

[0163] Furthermore, in an alternative but similar embodiment, theinvention provides a process for reducing an unsaturated organiccompound, comprising:

[0164] a) contacting H₂, at least one metal salt or complex, and atleast one nicotinamide cofactor to form at least some reducednicotinamide cofactor, and

[0165] b) contacting the reduced nicotinamide cofactor, a nicotinamidecofactor dependent enzyme, and an unsaturated organic compound underconditions effective to form at least some of the reduced organicproduct,

[0166] wherein

[0167] i) when the metal salt or complex is a platinum carbonyl clustercomplex, the catalyst does not comprise a redox active dye; and

[0168] ii) when the metal salt or complex is a rhodium phosphinecomplex, the nicotinamide cofactor dependent enzyme is not a mixture ofhorse liver alcohol dehydrogenase and lactate dehydrogenase.

[0169] In the embodiment described immediately above, the contactingsteps may occur simultaneously, or sequentially; the reaction steps mayalso occur in the same phase or reactor. Alternatively, the metal saltor complex, and the nicotinamide cofactor might be contacted with H₂ inone reaction phase or reactor, while the unsaturated organic product wascontacted with enzyme and the reduced organic cofactor in a differentphase or reactor.

[0170] In preferred embodiments of step a) of the process describedimmediately above, the H₂ reacts with the metal salt or complex to format least some of a metal hydrogen complex, and the metal hydrogencomplex reacts directly with the nicotinamide cofactor to transferhydrogen from the metal hydrogen complex to the nicotinamide cofactor.

[0171] In yet another aspect, the current invention provides acomposition for reducing unsaturated organic compounds comprising H₂ anda catalyst, the catalyst comprising:

[0172] a) at least one metal salt or complex,

[0173] b) at least one nicotinamide cofactor; and

[0174] c) a nicotinamide cofactor dependent enzyme,

[0175] wherein

[0176] i) when the metal salt or complex is a platinum carbonyl clustercomplex, the catalyst does not comprise a redox active dye; and

[0177] ii) when the metal salt or complex is a rhodium phosphinecomplex, the nicotinamide cofactor dependent enzyme is not a mixture ofhorse liver alcohol dehydrogenase and lactate dehydrogenase.

[0178] As previously described hereinabove, many embodiments of theabove-described processes for reducing unsaturated organic compoundswith H₂ employ oxidoreductase enzymes whose activity is enhanced ormaintained by the presence of certain stabilizers. It is known in theart to employ certain sulfur containing stabilizers, and in particularit is known in the art to employ stabilizers having sulfhydril groups,which are exemplified by compounds such as dithiothreitol (DTT),mercaptoethanol, and the like.

[0179] Unexpectedly, it has been discovered that certain phosphoruscontaining compounds can also stabilize oxidoreductase enzymes. Theenzyme stabilization occurs independently of the presence or absence ofthe H₂, or unsaturated organic compounds employed in the processesdescribed above. In particular, it has been unexpectedly discovered thatphosphines or phosphites can stabilize oxidoreductase enzymes in avariety of processes.

[0180] Therefore, in one aspect the invention provides a process forstabilizing the activity of an oxidoreductase enzyme, comprising mixinga phosphine or phosphite with an oxidoreductase enzyme. In preferredembodiments of the invention, the mixing of the phosphine or phosphitewith the oxidoreductase enzyme occurs in a liquid medium. The liquidmedium may comprise water, an organic solvent, or a mixture thereof.Preferably, the liquid medium comprises a substantially aqueous buffersolution.

[0181] Preferably, the mixing of the phosphine or phosphite with theoxidoreductase enzyme is effective to slow the loss of activity of theoxidoreductase enzyme, as compared to the rate of loss of activity ofthe oxidoreductase enzyme in the absence of the phosphine or phosphite.While not wishing to be bound by theory, it is believed that thephosphine or phosphite compounds serve as reducing agents for certainoxidation sensitive components of the enzymes, including cysteine aminoacid residues contained in the enzymes.

[0182] Preferably, the phosphine or the phosphite comprise phosphorusderivatives PR_(n)(O—R)_(3−n), wherein n is an integer of from zero tothree. Preferably, the R groups comprise from 1 to about 10 carbonatoms. The R groups (whether bonded to phosphorus or oxygen) areindependently selected, and may be the same or different. Preferably,the R groups are hydrocarbyl, or substituted hydrocarbyl, groups, whichinclude alkyl, cycloakyl, aromatic and heteroaromatic groups.

[0183] Preferably, one or more of the R groups is substituted with oneor more polar groups. Preferred polar groups include salts of anionicgroups such as sulfonate, and carboxylate groups; salts of cationicgroups such as alkylammonium groups, or polar but electrically neutralgroups such as hydroxyl, amino, alcohol, or polyalkylene glycol groups.Polyethylene glycol groups are preferred polyalkylene glycol groups. Thepolar groups are believed to be beneficial in improving theeffectiveness of the phosphine or phosphite stabilizers because thepolar groups tend to increase the water solubility of the phosphines orphosphites, so that they can more effectively interact with theoxidoreductase enzymes, which are also water soluble.

[0184] Experimental

[0185] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow the compounds, and associated processes and methods are constructed,used, and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.) but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. (Celsius)or is at ambient temperature, and pressure is at or near atmospheric.

[0186] Materials and Methods

[0187] Tris(m-sulfonatophenyl)phosphine trisodium salt (TPPTS) waspurchased from Strem Chemical and sodiumdiphenylphosphinobenzene-3-sulfonate (TPPMS) from TCI America. Alcoholdehydrogenase enzymes of Thermanaerobium brockii (TBADH, EC 1.1.1.2),β-nicotinamide adenine dinucleotide (NAD⁺), β-nicotinamide adeninedinucleotide phosphate (NADP⁺) and their respective reduced forms werepurchased from Sigma Chemical and stored in a refrigerator (−10° C.).The preparation of TBADH is described in U.S. Pat. No. 4,352,885.RuCl₃-hydrate (38.4% Ru) was obtained from Colonial Metals, Inc.1,3,5-Triaza-7-phosphaadamantane (PTA) was prepared by the procedure ofDaigle, et al. (Inorg. Synth. 1998, 32, 40-45). [Cl₂Ru(TPPTS)₂]₂ wasprepared by the procedure of Hernandez, and Kalck (J. Mol. Catal. A.Chem., 1993, 116, 117-130). Cl₂Ru(PTA)₄ was prepared by the procedure ofDarensbourg, et al., (Inorg. Chem., 1994, 33, 200-08). Phosphate bufferwas prepared using KHPO₄. Procedures for 2-heptanone reduction usingisopropanol to regenerate NADP were adapted from Keinan, et al. (J. Am.Chem. Soc. 1986, 108, 162). ¹H and ¹³C NMR spectra were recorded on aVarian Gemini-300 spectrometer. Reference samples of NAD⁺, NADH, NADP⁺and NADPH were dissolved in D₂O and their chemical shifts (δ) arereported relative to sodium 3-(trimethylsilyl)propionate (TMSP).Chemical shift assignments have been made in accord with Ragg, et al.(J. Biochim. Biophys. Acta 1991, 1076, 49) and Oppenheimer (Proc. Natl.Acad. Sci. U.S. 1971, 68, 3200). GLC was conducted on a HP 6890 gaschromatograph with a 30′ DB-FFAP capillary column and flame ionizationdetection. Samples of (R)-(−)-2-heptanol (95%) and (S)-(+)-2-heptanol(99%, 97% ee) were obtained from Aldrich Chemical. Samples were analyzedby conversion to their trifluoroacetate esters by a standardizedtreatment with trifluoroacetic anhydride. The enantiomeric excess of(S)-2-heptanol produced by enzymatic reduction was determined by chiralGC and comparison to (R)- and (S)-2-heptanol standards using aCyclodex-B column (30 m ×0.25, 0.25μ film) at 40° C. Under theseconditions the retention times of the trifluoroacetates of(R)-2-heptanol and (S)-2-heptanol were 28.15 minutes and 28.77 minutes.

EXAMPLE 1

[0188] Example 1 demonstrates that H₂ can be used to convert 2-butanoneto 2-butanol in the presence of a catalyst comprising a rutheniumcomplex, TBADH and NADP⁺.

[0189] NADP⁺ (28.0 mg, 36.6 μmol) was placed in 2 mL of a 0.1 Mphosphate buffer and the pH adjusted to 8.1 with NaOH. TBADH (1.2 mg,8.8 units) was added to this solution and it was placed in an 80 mLFisher-Porter bottle. The Fisher-Porter bottle was evacuated andrefilled with argon three times then [Cl₂Ru(TPPTS)₂]₂ (6.4 mg, 5.0 μmol)and 2-butanone (5 μl) were added under a flow of argon. TheFisher-Porter bottle was evacuated and filled with 70 psi of H₂ andheated to 40° C. for 2.0 hr. The pressure was released from the bottleand a liquid sample analyzed by GLC showed the production of 9.6 μmol of2-butanol.

Comparative Example 1a

[0190] Comparative Example 1a demonstrates that without the enzyme(TBADH) the conditions applied in Example 1 produce no reaction.

[0191] NADP⁺ (39.0 mg, 51.5 μmol) was placed in 2 mL of a 0.1 Mphosphate buffer. The pH of this solution was adjusted to 8.1 with NaOHand then placed in an 80 mL Fisher-Porter bottle. The Fisher-Porterbottle was evacuated and refilled with argon three times then[Cl₂Ru(TPPTS)₂]₂ (8.8 mg, 6.9 μmol) and 2-butanone (10 μl) were addedunder a flow of argon. The Fisher-Porter bottle was evacuated and filledwith 70 psi of H₂ and heated to 40° C. for 2.0 hr. The pressure wasreleased from the bottle and a liquid sample analyzed by GLC showed theproduction of no 2-butanol. The reaction solution was stripped todryness under reduced pressure and the residue was dissolved in D₂O. ¹HNMR analysis showed that only a trace of NADP⁺ remained and that NADPHwas the dominant species remaining.

EXAMPLE 2

[0192] Example 2 demonstrates that H₂ can be used to convert 2-heptanoneto 2-heptanol in significant enantiomeric in the presence of a catalystcomprising a ruthenium complex, TBADH and NADP⁺.

[0193] An 80 mL Fisher-Porter bottle with a micro-liquid sampling tubewas charged with 20 mL of a 0.1 M phosphate buffer (pH=7.0), 2-heptanone(100 μl, 718 μmol), TPPTS(74 mg, 130.7 μmol), NADP⁺ (10 mg, 13.1 μmol)and TBADH (2.7 mg, 19.8 units) under a flow of argon. Rutheniumcatalyst, [Cl₂Ru(TPPTS)₂]₂ (17 mg, 6.5 μmol), was finally added and theFisher-Porter bottle was sealed, evacuated and filled with 70 psi of H₂.Evacuation and refilling with H₂ were repeated two times and theapparatus was then heated to 60° C. with an oil bath. After 10 hours GCanalysis of a microsample removed via the liquid sampler showed theproduction of 227.8 μmol of 2-heptanone. After an additional four hoursan additional 13.0 μmol had accumulated (240.8 μmol). The pressure wasreleased from the bottle and a liquid sample analyzed by chiral GCshowed the 2-heptanol to be 70.5% (S) and 29.5% (R) (41.0% ee).Turnovers of catalyst components: NADP⁺ _(TO)=18.4

EXAMPLE 3

[0194] Example 3 demonstrates that the ruthenium catalyst[Cl₂Ru(TPPTS)₂]₂ is a catalyst precursor for the reduction of NAD(P)⁺with H₂.

[0195] NADP⁺ (36.0 mg, 47.0 μmol) was placed in 2 mL of a 0.1 Mphosphate buffer. The pH of this solution was adjusted to 8.3 with NaOHand then placed in an 80 mL Fisher-Porter bottle. The Fisher-Porterbottle was evacuated and refilled with argon three times then[Cl₂Ru(TPPTS)₂]₂ (9.7 mg, 3.7 μmol) was added under a flow of argon. TheFisher-Porter bottle was evacuated and filled with 70 psi of H₂ andheated to 40° C. for 3.0 hr. The volatiles were removed under reducedpressure and a sample dissolved in D₂O. To this was added 20 μl oftrimethylsilylpropionate standard solution. ¹H NMR integration showsthat 29.0 μmole of NADPH have been produced under these conditions.

[0196] NADP⁺

[0197]¹H (D₂O): δ 9.35 (s) N₂, 9.17 (d, 0.6 Hz) N₆, 8.84 (d, 1.0) N₄,8.42 (s) A₂, 8.21 (t, 0.7) N₅, 8.11 (s) A₈, 6.10 (d, 5.2 Hz) N_(1′),6.02 (d,) A_(1′), 4.77 (t,) A_(2′), 4.77 (t,) A_(2′), 4.56 (br s),4.51(br s), 4.45 (br s), 4.40 (br s), 4.26(br s).

[0198] NADPH

[0199]¹H (D₂O): δ 8.48 (s) A₈, 8.21 (s) A₂, 6.94 (s) N₆, 6.13 (d,)A_(1′), 5.98 (d, 0.7) N_(1′), 8.11 (s) A₈, 6.10 (d, 5.2 Hz) N_(1′), 6.02(d,) A_(1′), 4.78 (t,) A_(2′), 4.71 (t,) A_(2′), 4.51 (t,), 4.39 (br s),4.22 (br m), 4.09 (br s), 2.71 (dd,) P₄

EXAMPLE 4

[0200] Example 4 demonstrates that the ruthenium catalyst[Cl₂Ru(TPPTS)₂]₂ is a catalyst precursor for the reduction of NAD⁺ withH₂.

[0201] NAD⁺ (34.0 mg, 38.0 μmol) was placed in 2 mL of a 0.1 M phosphatebuffer. The pH of this solution was adjusted to 8.1 with NaOH and thenplaced in an 80 mL Fisher-Porter bottle. The Fisher-Porter bottle wasevacuated and refilled with argon three times then [Cl₂Ru(TPPTS)₂]₂ (6.9mg, 2.6 μmol) was added under a flow of argon. The Fisher-Porter bottlewas evacuated and filled with 70 psi of H₂ and stirred to 23° C. for17.0 hr. The volatiles were removed under reduced pressure and a sampledissolved in D₂O To this was added 20 μl of trimethylsilylpropionatestandard solution. ¹H NMR integration shows that 9.2 μmole of NADH havebeen produced under these conditions.

[0202] NAD⁺

[0203]¹H(D₂O): δ 9.35 (s) N₂, 9.17 (d, 0.6 Hz) N₆, 8.84 (d, 1.0) N₄,8.42 (s) A₂, 8.21 (t. 0.7) N₅, 8.11(s) A₈, 6.10 (d, 5.2 Hz) N_(1′), 6.02(d,) A_(1′), 4.77 (t,) A_(2′), 4.56 (br s), 4.51 (br s), 4.45 (br s),4.40 (br s), 4.26 (br s).

[0204] NADH

[0205]¹H(D₂O): δ 8.48 (s) A₈, 8.21 (s) A₂, 6.94 (s) N₆, 6.13 (d,)A_(1′), 5.98 (d, 0.7) N_(1′), 8.11 (s) A₈, 6.10 (d, 5.2 Hz) N_(1′), 6.02(d,) A_(1′), 4.78 (t,) A₂, 4.71 (t,) A_(2′), 4.51 (t,), 4.39 (br s),4.22 (br m), 4.09 (br s), 2.71 (dd,) P₄.

EXAMPLE 5

[0206] Example 5 demonstrates that iodide is not a poison for thecatalyst derived from ruthenium complex [Cl₂Ru(TPPTS₎₂]₂.

[0207] NAD⁺ (34.0 mg, 38.0 μmol) was placed in 2 mL of a 0.1 M phosphatebuffer containing 0.07 M sodium iodide. The pH of this solution wasadjusted to 8.1 with NaOH and then placed in an 80 mL Fisher-Porterbottle. The Fisber-Porter bottle was evacuated and refilled with argonthree times then [Cl₂Ru(TPPTS)₂]₂ (7.7 mg, 2.9 μmol) was added under aflow of argon. The Fisher-Porter bottle was evacuated and filled with 70psi of H₂ and stirred to 23° C. for 13.0 hr. The volatiles were removedunder reduced pressure and a sample dissolved in D₂O. To this was added20 μl of trimethylsilylpropionate standard solution. ¹H NMR integrationshows that 7.4 μmole of NADH have been produced under these conditions.

EXAMPLE 6

[0208] Example 6 demonstrates that other water-soluble rutheniumcomplexes are catalysts for cofactor regeneration with H₂.

[0209] Using a procedure identical to that of Example 4 aboveCl₂Ru(PTA)₄ (4.8 mg, 6.0 μmol) and NAD⁺ (33.0 mg, 50.0 μmol) werereacted at 70 psi and 40° C. for 15.5 hr. ¹H NMR assay shows that 11.0μmole of NADH have been produced under these conditions.

EXAMPLE 7

[0210] Example 7 demonstrates that the phosphine ligands used in theabove examples in ruthenium complexes are necessary to generatecatalysts for cofactor regeneration.

[0211] Using a procedure identical to that of Example 4 aboveRuCl₃-hydrate (1.6 mg, 38.4% Ru, 6.0 μmol) and NAD⁺ (33.0 mg, 50.0 μmol)were reacted at 70 psi and 40° C. for 2.5 hr. ¹H NMR assay shows that noNADH has been produced under these conditions.

EXAMPLE 8

[0212] Example 8 demonstrates that the ruthenium complexes used in theabove examples are necessary for the regeneration of cofactor withhydrogen.

[0213] Following the procedure of Example 4, but excluding the rutheniumcatalyst, NAD⁺ (33.0 mg, 50.0 μmol) was placed 2 mL of a 0.1 M (aq)KHPO₄ with 0.07 M sodium iodide in an 80 mL Fisher-Porter bottle and thepH adjusted to 8.15 with NaOH. After establishing a H₂-atmosphere (70psig) and heating to 40° C. for 2.5 hr ¹H NMR analysis showed noproduction of NADH.

EXAMPLE 9

[0214] Example 9 demonstrates that the hydrogen is necessary for theregeneration of cofactors described in the above examples.

[0215] Following the procedure of Example 4, [Cl₂Ru(TPPTS)₂]₂ (7.9 mg,6.0 μmol) and NAD⁺ (33.0 mg, 50.0 μmol) was placed 2 mL of 0.1 M (aq)KHPO₄ with 0.07 M sodium iodide and an atmosphere of argon rather thanH₂, was placed over the reaction solution. After heating to 40° C. for2.5 hr. ¹H NMR analysis showed no NADH. The argon atmosphere was thenreplaced by H₂ and NADH was produced without significant catalystdeactivation.

EXAMPLE 10

[0216] Example 10 demonstrates that the TPPTS can be used as astabilizer for TBADH.

[0217] Phosphate buffer (10 mL, 0.1 M, pH=7.0) was placed in a 100 mLSchlenk tube under argon. 2-Heptanone (4.92 g, 43.1 mmol), isopropanol(1.57 g, 26.1 mmol), heptadecane (41.0 μl), TPPTS (23 mg, 40.0 μmol),NADP⁺ (0.4 mg, 0.5 μmol) and TBADH (2.7 mg, 19.8 units) were then addedunder a flow of argon. After evacuation and refilling with argon threetimes the apparatus was heated to 38° C. with an oil bath. GC analysisevery five hours showed a continuous production of 2-heptanol for theinitial 20 hours at a rate corresponding to˜210 turnovers of NADP⁺ perhour (total of 2.17 mmoles of 2-heptanol in 21 hours). After andadditional 24 hours the yield of 2-heptanol had not changed. The finalyield of 2-heptanol corresponds to 4,343 turnovers of NADP⁺.

Comparative Example 10a

[0218] Comparative Example 10a shows TBADH productivity without astabilizer.

[0219] Identical amounts of all reagents in a 2-heptanone reduction withisopropanol (Example 10) except for the water-soluble phosphine (TPPTS)were heated to 38° C. with an oil bath for 23 hours. The yield of2-heptanol was 0.13 mmol, corresponding to a total of 271 turnovers ofNADP⁺.

Comparative Example 10b

[0220] Comparative Example 10b shows TBADH productivity withdithiothreitol (DTT) as a stabilizer.

[0221] Using the procedure described in Comparative Example 10a,identical amounts of all reagents except twice the amount of 2-heptanone(9.84, 86.2 mmol) and substituting DTT as a stabilizer (6 mg, 40 μmol)in place of TPPTS, the reduction of 2-heptanone with isopropanol wascarried out at 38° C. for 19 hours yielding 2.51 mmol of 2-heptanol,corresponding to a total of 5,021 turnovers of NADP⁺.

Comparative Example 10c

[0222] Comparative Example 10c shows TBADH productivity withmercaptoethanol as a stabilizer.

[0223] Using the procedure described in Example 10 and identical amountsof all reagents but substituting mercaptoethanol as a stabilizer (2.8μl, 40 μmol) in place of DTT, the reduction of 2-heptanone withisopropanol was carried out at 38° C. for 22 hours yielding 2.56 mmol of2-heptanol, corresponding to a total of 5,121 turnovers of NADP⁺.

EXAMPLE 11

[0224] Example 11 demonstrates that triphenylphosphine (TPP) can be usedas a stabilizer for TBADH.

[0225] Using the procedure described in Example 10 and identical amountsof all reagents but substituting triphenylphosphine as a stabilizer (10mg, 40 μmol) in place of TPPTS, the reduction of 2-heptanone withisopropanol was carried out at 37° C. for 19 hours yielding 2.15 mmol of2-heptanol, corresponding to a total of 4,301 turnovers of NADP⁺.

EXAMPLE 12

[0226] Example 12 demonstrates that “monosulfonated triphenylphosphine”(TPPMS) can be used as a stabilizer for TBADH.

[0227] Using the procedure describe in Example 10 and identical amountsof all reagents but substituting triphenylphosphine as a stabilizer (15mg, 40 μmol) in place of TPPTS, the reduction of 2-heptanone withisopropanol was carried out at 38° C. for 16 hours yielding 1.93 mmol of2-heptanol, corresponding to a total of 3,859 turnovers of NADP⁺.

[0228] Throughout this application, various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference into this application.

[0229] It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 32. A process for stabilizing the activity of an oxidoreductase enzyme, comprising mixing a phosphine or phosphite with an oxidoreductase enzyme.
 33. The process of claim 32, wherein the phosphine or the phosphite comprise phosphorus derivatives PR_(n)(O—R)_(3−n), wherein n is an integer from zero to three, the R groups may be the same or different, and the R groups comprise hydrocarbyl, substituted hydrocarbyl, aromatic or heteroaromatic groups.
 34. The process of claim 33, wherein the one or more R groups are substituted with one or more polar groups.
 35. The process of claim 34, wherein the polar group comprises sulfonate, carboxylate, amino, alkylammonium, hydroxyl, or polyalkylene glycol groups.
 36. The process of claim 32, wherein the mixing occurs in a liquid medium.
 37. The process of claim 32, wherein the oxidoreductase enzyme is an enzyme characterized under the EC classification system as having a first digit EC classification of
 1. 38. The process of claim 32, wherein the oxidoreductase enzyme is a nicotinamide cofactor dependent enzyme.
 39. The process of claim 32, wherein the oxididoreductase enzyme comprises a cysteine amino acid residue.
 40. The process of claim 32, wherein the rate of loss of activity of the oxidoreductase enzyme is slower in the presence of the phosphine or phosphite as compared to the rate of loss of activity of the oxidoreductase enzyme in the absence of the phosphine or phosphite.
 41. The process of claim 36, wherein the liquid medium comprises water, an organic solvent, or a mixture thereof.
 42. The process of claim 36, wherein the liquid medium is a substantially aqueous buffer solution.
 43. The process of claim 32, wherein the phosphine or phosphite compound has one or more anionic sulfonate groups.
 44. The process of claim 32, wherein the phosphine compound is a salt of tris(m-sulfonatophenyl)phosphine.
 45. The process of claim 32, wherein the phosphine or phosphite is a water soluble phosphine or phosphite. 